LOCUS Exported 6217 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination. May not contain entire coding region of gene. ACCESSION . VERSION . KEYWORDS 6MF9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6217) TITLE SGC Toronto Q3-Q4 2018 REFERENCE 2 (bases 1 to 6217) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6217 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 91..108 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 121..141 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(718..736) /label=T7 Term /note="T7 terminator, reverse primer" terminator 732..779 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(954..982) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(1009..1027) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1095..1199 /gene="bla" /label=AmpR promoter CDS 1200..2060 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1418..1437) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2231..2819 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2720..2739 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2973..2990 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3005..3144 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3070..3089) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3230..3252 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3246..3437) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4749..5831) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 5793..5812 /label=LacI-R /note="LacI, reverse primer" promoter complement(5832..5909) /gene="lacI" /label=lacI promoter primer_bind 6115..6134 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atataccatg catcatcatc atcatcacag cagcggcaga 121 gaaaacttgt atttccaggg cttaaatggt ggctcccaga catgtaactt taataatgct 181 ctaaatcctt caaacggagg agctggctta catatttcaa ataacttgga tggatcccat 241 aacgtaatta atgcaggacc taatattacc tcctacactg aagctttaga tgaattttgt 301 attgaacttc aaagaattat taatagtaca aaaactcttc atcactattc acatgtattt 361 tggaataggg ttagtgaaag aattgcacca aattattata atttagttaa aaggccaatg 421 tggcttcaat taatgataaa taaatgcaaa aaaagggaat acaaatctag aaaagatttc 481 caagatgact tggacttaat agttgagaac tgcaagattt ataatggggt aaatcatcct 541 cttgtatctg ttgcaacctt aatacattca aatgttgtaa aaaagattga tgagattcaa 601 ggaattgaaa agattgaagc ttatttatca ctaaaaccct gatgacgaag cttgcggccg 661 cactcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg 721 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc 781 tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa 841 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga 901 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag 961 cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac 1021 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 1081 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1141 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 1201 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 1261 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 1321 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 1381 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 1441 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 1501 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 1561 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 1621 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 1681 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 1741 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 1801 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 1861 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 1921 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 1981 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 2041 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 2101 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 2161 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 2221 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 2281 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 2341 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 2401 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 2461 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 2521 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 2581 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 2641 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 2701 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 2761 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 2821 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 2881 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 2941 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3001 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg 3061 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg 3121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga 3181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 3241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca 3301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg 3361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt 3421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg 3481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg 3541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa 3601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc 3661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt 3721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac 3781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca 3841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc 3901 cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac 3961 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga 4021 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg 4081 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg 4141 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg 4201 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa 4261 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc 4321 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga 4381 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct 4441 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc 4501 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga 4561 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca 4621 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 4681 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg 4741 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4801 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc 4861 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc 4921 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc 4981 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca 5041 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca 5101 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg 5161 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga 5221 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac 5281 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 5341 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc 5401 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta 5461 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg 5521 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat 5581 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca 5641 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc 5701 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc 5761 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt 5821 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg 5881 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc 5941 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa 6001 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 6061 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 6121 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 6181 tccggcgtag aggatcgaga tctcgatccc gcgaaat //